Effects of flt-3 ligand in combination with TPO on the expansion of megakaryocytic progenitors

As an early acting growth factor, flt-3 ligand (FL) promotes the ex vivo expansion of hematopoietic stem and progenitor cells. The effect and mechanism of FL on the development of the megakaryocytic lineage remain unclear. In this study, we compared the effects of FL and stem cell factor (SCF) in combination with other megakaryocyte-promoting cytokines on the differentiation and proliferation of megakaryocytic progenitors and investigated the expression of flt-3 receptors on megakaryocytic cell lines. In liquid cultures of enriched CD34+ cells from human umbilical cord blood for 14 days, FL plus thrombopoietin (TPO), interleukin-3 (IL-3), and IL-6 promoted the expansion of nucleated cells, CD34+ cells, CD34+ CD38- cells, and megakaryocyte colony-forming units (CFU-MK) by 300 +/- 115-, 23.8 +/- 11.3-, 33.9 +/- 28.6-, and 584 +/- 220-fold, respectively. Replacing FL with SCF significantly decreased the yield of all cell types. Using murine bone marrow (BM) cells, we demonstrated that FL at a range of 0-100 ng/ml had no significant mitogenic effect on CFU-MK formation. TPO increased CFU-MK (p < 0.001) but the effect was not significantly modified by FL. While one human acute lymphoblastic leukemia sample expressed high levels of flt-3 receptor, the four megakaryocytic cell lines (Meg-01, CHRF-288-11, M-07e, and Dami) did not show any positive expression. Our data suggest that the present cytokine combination and expansion conditions provide an effective and potentially useful system for the clinical expansion of cord blood for bone marrow transplantation (BMT). FL alone did not stimulate megakaryocytopoiesis, possibly due to the lack of receptor expression on megakaryocytes. The effect of FL in augmenting the expansion of CFU-MK in liquid culture might be due to the early action of FL at the pluripotent stem cell stage.     

人脐血造血干/祖细胞的磁力搅拌悬浮培养及移植实验

目的 探讨磁搅拌大规模培养体系对人脐血造血祖细胞的扩增效果以及扩增的人造血祖细胞植入动物体内后的造血重建情况.方法 从新鲜抗凝脐血中分离出单个核细胞(MNC),以添加干细胞因子、酪氨酸激酶受体3配基及血小板生成素的无血清培养体系进行培养.静态扩增组的细胞置于T25培养瓶中培养,磁搅拌悬浮扩增组(磁搅拌扩增组)的细胞采用Celstir装置进行培养,培养体系为50～100 ml.培养7 d后进行细胞计数、集落培养检测和细胞表面分子表达的测定.以不进行培养者为对照组.非肥胖糖尿病重症联合免疫缺陷(NOD/SCID)小鼠在接受2.5 Gy的亚致死剂量X射线照射后分别从尾静脉输入上述静态扩增组、磁搅拌扩增组和对照组的MNC(5×106个),另设不移植的空白对照组.观察小鼠的存活情况,6周后处死存活小鼠,检测骨髓细胞中CD34+细胞、CD3+细胞、CD19+细胞、CD33+细胞及CD45+细胞的含量以及人特异的Cart-Ⅰ和Alu基因的表达.结果 经过7天的培养,磁搅拌扩增组的造血祖细胞扩增倍数为(2.8±0.45)倍,明显高于静态扩增组的(2.1±0.48)倍(P<0.01).磁搅拌扩增组形成的红系集落、粒-巨噬细胞集落数均明显高于静态扩增组(P<0.05).静态扩增组扩增后的CD34+细胞、CD34+CD38-细胞和CD133+细胞含量均高于磁搅拌扩增组(P<0.05),而CD184+细胞和CD62L+细胞含量低于磁搅拌扩增组(P<0.01).移植后6周,对照组、静态扩增组和磁搅拌扩增组分别有3、4、5只小鼠存活,三组间两两比较,6周存活率的差异无统计学意义(P>0.05).存活6周的小鼠,其骨髓中能检人特异性CD34+细胞,以及CD3+细胞、CD19+细胞、CD33+细胞及CD45+细胞,也检测到人Alu基因和Cart-Ⅰ基因的表达.结论 磁搅拌培养能大规模扩增脐带血造血祖细胞,扩增的细胞能植入x射线照射的NOD/SCID小鼠,并重建其多系造血.     

CD34+ selected bone marrow grafts are radioprotective and establish mixed chimerism in dogs given high dose total body irradiation.

BACKGROUND: Canine stem cell transplantation models have provided important preclinical information for human clinical studies. The recent cloning of cDNA for canine CD34 and the production of monoclonal antibodies that recognize canine CD34 have been the basis for the development of techniques for the large-scale enrichment of canine hematopoietic progenitor cells. In this study, we evaluated the in vivo functional properties of canine bone marrow CD34+ cells after a myeloablative conditioning regimen. METHODS: After 920 cGy total body irradiation, three dogs received infusion of autologous CD34+ selected cells from the marrow, three dogs CD34+ depleted autologous marrow cells, and two dogs received CD34+ autologous marrow cells that were immunomagnetically selected and then further purified by cell sorting. In addition, four dogs received allogeneic marrow enriched for CD34+ cells from dog leukocyte antigen-identical littermates to investigate long-term repopulating function of CD34+ cells. Chimerism studies were performed using polymerase chain reaction to detect highly polymorphic microsatellite markers. RESULTS: In three recipients of autologous marrow enriched for CD34+ cells to between 29% and 70% (1.6 x 10(6) to 3.4x10(6) CD34+ cells/kg), prompt and full hematopoietic recovery occurred, whereas in three dogs that received marrow depleted of CD34+ cells (1 x 10(7) cells/kg), no hematopoietic recovery was achieved. In two dogs that received highly purified CD34+ cells (purity: 98% and 96%, 0.79x10(6) to 0.547x 10(6) CD34+ cells/kg), delayed but full hematopoietic recovery was seen. Three of four allograft recipients of 1.75x10(6) to 6.8x10(6) CD34+ cells/kg engrafted and showed full hematopoietic recovery, whereas one dog rejected the graft. The three long-term survivors showed stable mixed hematopoietic chimerism with predominantly donor hematopoiesis. CONCLUSION: Transplantation of canine CD34+ cells after lethal total body irradiation provides radioprotection and gives rise to long-term hematopoietic reconstitution. Stable donor/host mixed chimerism was observed in allograft recipients most likely as a result of T-cell depletion of the grafts. Our findings suggest a future role for canine preclinical transplant studies involving in vitro manipulation of hematopoietic pro.     

Rapid isolation of human CD34 hematopoietic stem cells--purging of human tumor cells.

Human CD34+ hematopoietic stem cells were purified using a new technology in which monoclonal antibodies are covalently immobilized on polystyrene surfaces. The CD34+ cell isolation scheme involved three sequential processes: (1) purification of bone marrow mononuclear cells; (2) enrichment of CD34+ cells using covalently immobilized soybean agglutinin; and (3) positive selection of CD34+ cells using polystyrene surfaces coated with the anti-CD34 monoclonal antibody ICH3. CD34+ cells purified by this process have both low-to-medium forward light scatter and low 90 degrees light-scatter properties. Moreover, the purified CD34+ cells are greater than 85% viable, express appropriate characteristic surface antigens, and are 10-50-fold enriched in short- and long-term hematopoietic activity. CD34+ cells collected in this manner from bone marrow samples contaminated with radiolabeled breast carcinoma, neuroblastoma, acute myelogenous leukemia, or small cell lung carcinoma cells were 99.9% depleted of the tumor cells. The CD34+ cell selection devices are sterile and are easily scaled-up to process clinical scale bone marrow samples.     

Donor bone marrow cells play a role in the prevention of accelerated graft rejection induced by semi-allogeneic spleen cells in transplantation

Spleen or spleen plus bone marrow cells from (BALB/cxC57Bl/6)F1 donors were transferred into BALB/c recipients 21 days before skin or cardiac transplantation. Prolonged graft survival was observed on recipients treated with the mixture of donor-derived cells as compared to those treated with spleen cells alone. We evaluated the expression of CD45RB and CD44 by splenic CD4+ and CD8+ T cells 7 and 21 days after donor cell transfer. The populations of CD8+CD45RBlow and CD8+CD44high cells were significantly decreased in mice pre-treated with donor spleen and bone marrow cells as compared to animals treated with spleen cells only, although these cells expanded in both groups when compared to an earlier time-point. No differences were observed regarding CD4+ T cell population when recipients of donor-derived cells were compared. An enhanced production of IL-10 was observed seven days after transplantation in the supernatants of spleen cell cultures of mice treated with spleen and bone marrow cells. Taken together these data suggest that donor-derived bone marrow cells modulate the sensitization of the recipient by semi-allogeneic spleen cells in part by delaying the generation of activated/memory CD8+ T cells leading to enhanced graft survival.     

FLT3 ligand promotes engraftment of allogeneic hematopoietic stem cells without significant graft-versus-host disease

BACKGROUND: Graft-versus-host (GVH) reactions contribute to stable engraftment of allogeneic hematopoietic stem cell transplants. It was hypothesized that the in vivo expansion of recipient dendritic cells (DC) with the administration of ligand for Flt3 (FL) could promote allogeneic engraftment after reduced-intensity conditioning by enhancing the GVH effect. METHODS: FL was first administered to three nonirradiated healthy dogs for 13 days at a dosage of 100 microg/kg/day. Next, nine dogs received 4.5 Gy total-body irradiation (TBI) and unmodified marrow grafts from dog leukocyte antigen (DLA)-identical littermates without posttransplant immunosuppression. FL was administered to the recipients at a dosage of 100 microg/kg/day from day -7 until day +5. RESULTS: In normal dogs, FL produced significant increases in monocytes (CD14+) and neutrophils in the peripheral blood, a marked increase in CD1c+ cells with DC-type morphology in lymph nodes, and increased alloreactivity of third-party responders to peripheral blood mononuclear cells in mixed lymphocyte reactions (P<0.001). Sustained engraftment was observed in eight of nine (89%) FL-treated dogs compared with 14 of 37 (38%) controls (P=0.02, logistic regression). All engrafted FL-treated dogs became stable complete (n=2) or mixed (n=6) hematopoietic chimeras without significant graft-versus-host disease (GVHD). Recipient chimeric dogs (n=4) were tolerant to skin transplants from their marrow donors but rejected skin grafts from unrelated dogs within 7 to 9 days (median, 8 days). CONCLUSIONS: In this study, the authors showed that FL administered to recipients promotes stable engraftment of allogeneic marrow from DLA-identical littermates after 4.5 Gy TBI without significant GVHD.     

Ex vivo expansion of canine dendritic cells from CD34+ bone marrow progenitor cells

BACKGROUND: The aims of this study were to ex vivo expand canine dendritic cells and determine their phenotype and functional characteristics. METHODS: CD34+-selected cells and CD34+-depleted canine bone marrow (BM) cells were cultured in Iscove's modified medium for 14 days. Cytokines added to the cultures included human granylocyte/macrophage colony-stimulating factor 5 ng/ml, hFlt3 ligand 200 ng/ml, and human tumor necrosis factor-alpha 10 ng/ml. Cultured cells and purified subpopulations were assessed for cell surface antigen expression, morphology, and function by flow cytometric analysis, electron microscopy, and an allogeneic mixed lymphocyte reaction at day 14. RESULTS: Two main cell populations were identified, DR++(bright)/CD14- and DR+(dim)/CD14+. Ex vivo expanded CD34+-selected cells showed increased allostimulatory activity compared to both cultured CD34+-depleted cells and mononuclear cells. In contrast, ex vivo expansion from CD34+-depleted cells was unsuccessful. After sorting cells from the ex vivo expanded CD34+-selected bone marrow to enrich for DR++/CD14- cells, a 42-fold increase (median) of allostimulatory activity was observed as compared with sorted DR+/CD14+ cells (P=0.02). CONCLUSIONS: Cells with dentric cell-like phenotypes and functions can be cultured from canine CD34+-selected bone marrow cells. Future studies will address the roles of these cells in engraftment, graft versus host reactions and graft-host tolerance in a canine hematogoietic stem cell transplantaton model.     

Safety and efficacy of autologous bone marrow stem cell transplantation through hepatic artery for the treatment of chronic liver failure: a preliminary study

This study was performed to determine the safety and tolerability of injecting autologous bone marrow stem cells (BMC) (CD34+) into four patients with liver insufficiency. The study was based on the hypothesis that the CD34+ cell population in granulocyte colony stimulating factor (G-CSF) mobilized blood and autologous bone marrow contains a subpopulation of cells with the potential for regenerating damaged tissue. We separated the CD34+ stem cell population from the bone marrow. The potential of the BMC to differentiate into hepatocytes and other cell lineages has already been reported. Several reports have also demonstrated the plasticity of hematopoietic stem cells to differentiate into hepatocytes. Recently Sakaida demonstrated reduction in fibrosis in chemically induced liver cirrhosis following BMC transplantation. From a therapeutic point of view, chronic liver cirrhosis is one of the targets for BMC transplantation. In this condition, there is excessive deposition of extracellular matrix and hepatocyte necrosis. Encouraged by this evidence that the CD34+ cell population contains cells with the potential to form hepatocyte-like elements, four patients with liver insufficiency were given G-CSF to mobilize stem cells. CD34+ cells (0.1 x 10(8)) were injected into the hepatic artery. No complications or specific side effects related to the procedure were observed; four patients showed improvements in serum albumin, bilirubin and ALT after one month from the cell infusion.     

人脐血造血干/祖细胞的生物反应器大规模扩增及移植实验

目的 利用生物反应器大规模扩增人脐血造血干/祖细胞,并通过动物移植实验检验该方法的有效性.方法 采集抗凝脐血10份,分离出单个核细胞(MNC),分别进行生物反应器扩增培养和静态扩增培养.检测扩增前后细胞表面CD34、CD38、CD133、CD184和CD62L分子的表达,并进行造血干/祖细胞集落的培养.取非肥胖糖尿病重症联合免疫缺陷小鼠,以X射线照射后,分为4组,其中MNC组小鼠注射未经扩增培养的MNC;静态扩增组小鼠注射经过静态扩增培养的细胞;反应器扩增组小鼠注射经过生物反应器扩增培养的细胞;空白对照组小鼠注射生理盐水.移植后6周处死存活小鼠,收集骨髓细胞,检测其中CD45+、CD3+、CD19+和CD33+细胞的含量以及人特异的Cart-Ⅰ和Alu基因的表达.结果 生物反应器扩增前MNC为(1.2～2.8)×108个,扩增后为(3.7～12.6)×108个,扩增后的细胞数明显高于静态扩增培养者(P<0.01).经生物反应器扩增后所形成的红系集落形成单位、粒-巨噬细胞集落形成单位数明显高于经静态扩增者(P<0.05).移植6周后,空白对照组小鼠均死亡,MNC组存活率为35%,静态扩增组存活率为30%,反应器扩增组存活率为62.9%,后者明显高于前二者(P<0.05).各组存活小鼠骨髓细胞中均检测到Alu基因和Cart-Ⅰ基因的表达以及人源CD33+、CD45+、CD3+及CD19+细胞.结论 利用生物反应器可大规模扩增人脐血造血干/祖细胞,所得细胞能植入小鼠体内,并能获得造血功能重建.     

The xenotransplantation of goat and human hematopoietic cells to sheep fetuses

Hematopoietic xenografts were carried out in three experiments using goat fetal liver (44-48 days, experiments I and II) or purified human CD 34+ cells (experiment III) as the donor cells. Recipients were sheep fetuses at 41-47 days of gestation. Goat fetal liver cells were either injected without any pretreatment or stimulated by preincubation in a culturing in goat phytohemagglutinin-stimulated lymphocyte supernatant. Human CD 34+ myeloid progenitor cells were purified from bone marrow by minimacs immunomagnetic purification and cultured in medium supplemented with stem cell factor, IL3, and IL6. Goat-sheep chimerism was assessed by flow cytometry analysis (FCA) of peripheral blood and bone marrow cells using a mouse anti-goat CD 45 monoclonal antibody and by karyotype analysis of peripheral blood from goat/sheep chimeras. Human cell engraftment was assessed by polymerase chain reaction amplification of the human DAX1 gene in blood and bone marrow DNA from sheep which had received human cells. In the three experiments, a mean of 76% (26 of 34) of injected fetuses were born alive without any clinical evidence of graft-versus-host disease. Three lambs were found to be goat/sheep chimeric after flow cytometry analysis (peripheral blood and bone marrow) and karyotype (peripheral blood) analysis. Both tissues continued to express goat cells at 6 or 12 months (last assessment) depending on the experiment. No human chimerism was detected using polymerase chain reaction amplification in peripheral blood and bone marrow of any of the six sheep grafted with human cells. These data and those also obtained on other species (human, pig/sheep) show that it is possible to carry out hematopoietic xenografts using the sheep fetus as recipient provided both donor and recipient fetal cells are processed during the period of tolerance to foreign antigens.     

The effect of cryopreservation on clonogenic capacity and in vitro expansion potential of umbilical cord blood progenitor cells

BACKGROUND: Umbilical cord blood progenitor cells have been demonstrated to possess significant advantages over bone marrow in terms of proliferative capacity and immunologic reactivity. But the low number of hematopoeitic stem cells (HSC) is the most important limitation of its use. The ex vivo expansion of cord blood progenitor cells is the current strategy to overcome this problem. Furthermore, among the factors that enable successful cord blood transplantation is the ability to store and subsequently recover a sufficient number of viable cells. Since it would be costly to expand umbilical cord blood (UCB) progenitor cells, it is important to determine the feasibility and reproducibility of progenitor cell expansion after cryopreservation. We evaluated whether cryopreservation procedures might impair the clonogenic capacity and in vitro expansion of UCB. MATERIALS AND METHODS: We evaluated the cell viability, clonogenic capacity, CD34+38- content and in vitro expansion potential of progenitor cells from UCB (n = 10) separated mononuclear cells (MNC), before and after 1 month of cryopreservation by programmed rate freezing. RESULTS: Although cell viability decreased after cryopreservation (P < .05), there was no significant difference in CD34+ or CD34+38- absolute count, colonogenic capacity and in vitro expansion potential of cord blood progenitor cells (P > .05). CONCLUSIONS: Since the survival of CD34+ cells was greater than other elements, CD34+ cells seem more tolerant to cryopreservation than the other nucleated populations. Moreover in vitro expansion of UCB progenitor cells may be obtained following cryopreservation. Our results suggest that cryopreservation procedures do not impair the clonogenic capacity and in vitro expansion potential of cord blood stem/progenitor cells.     

动员自体骨髓干细胞对大鼠急性肾小管坏死的治疗

目的：观察粒细胞集落刺激因子联合干细胞因子动员骨髓干细胞的作用、骨髓干细胞是否具有向损伤肾组织归巢的能力及其在肾脏组织中的分布,初步探讨粒细胞集落刺激因子联合干细胞因子是否具有促进急性肾小管坏死修复的作用。方法：160只8～10周龄雄性SD大鼠随机分为4组：对照组,模型组、G-CSF＋SCF治疗组、G-CSF＋SCF对照组,检测：（1）外周血白细胞总数及单个核细胞中CD34＋细胞百分比的变化;（2）尿NAG酶检测;（3）肾脏组织病理学改变;（4）肾组织CD34＋细胞表达变化。结果：（1）G-CSF＋SCF治疗组和G-CSF＋SCF对照组外周血中白细胞数、CD34＋细胞百分比于第5天达高峰,与对照组、模型组相比,差异有统计学意义（P〈0.05）,以后逐渐下降;相应地,G-CSF＋SCF治疗组肾组织内CD34＋细胞较对照组、模型组也明显增多（P〈0.05）。（2）手术后第5、10、17天,G-CSF＋SCF治疗组尿NAG酶、肾脏病理学改变均明显好于模型组（P〈0.05）。第24天G-CSF＋SCF治疗组尿NAG酶、肾脏病理学改变基本恢复正常,而模型组仍异常。第31天各组间尿NAG酶、肾脏病理学改变其差异无统计学意义。结论：（1）粒细胞集落刺激因子和干细胞因子联合应用对缺血再灌注损伤诱发急性肾小管坏死大鼠的骨髓干细胞有显著的动员作用。（2）骨髓干细胞能在损伤的肾小管归巢和定居,并可能参与损伤肾组织的修复。（3）粒细胞集落刺激因子和干细胞因子联合应用能在一定程度上加速急性肾小管坏死后肾功能的修复。     

乙肝肝移植受体术后骨髓CD34+细胞和PBMCs内乙肝病毒DNA的同步检测

目的 肝移植术后乙肝复发严重影响预后,HBV感染的病毒来源值得研究.采用PCR技术可检测到术后病人外周血单个核细胞(PBMCs)中HBV DNA的存在,然而PBMCs的寿命有限,其中的病毒来源仍不清楚.该研究对此问题做了初步的探索.方法 采集13例乙肝肝移植术后受体外周血和骨髓标本,运用Ficoll密度梯度离心法结合免疫磁性分离法(MACS)分别从外周血和骨髓标本中分离出PBMCs和骨髓来源CD34+细胞即造血干祖细胞,利用实时荧光定量PCR法检测细胞中的HBV DNA,同时检测血清HBV标志物和血清HBV DNA.结果 13例病人术前血清HBV DNA阳性8例,阴性5例.术后平均37个月的随访期内(16～77个月)血清中HBV DNA,HBsAg和HBeAg检测结果全为阴性;13例PBMCs中均检测到HBV DNA的存在,阳性率为100%.PBMCs中DNA含量对数平均值为3.40±0.85;13例骨髓来源CD34+细胞中都检测到HBV DNA的存在.阳性率也是100%.CD34+细胞中DNA含量对数平均值为3.30±0.58.病人PBMCs和CD34'细胞内DNA含量对数值均数差异无统计学意义(P>0.05).同一病人PBMCs与CD34+细胞HBV DNA同时为阳性.术前血清HBV DNA检测阳性和阴性病人的PBMC内DNA含量对数值均数差异无统计学意义(P＞0.05);术前血清HBV DNA检测阳性和阴性病人的CD34+细胞内DNA含量对数值均数差异也无统计学意义(P＞0.05).结论 现有预防措施下,乙肝肝移植受体虽然外周血清中不能检测到HBV DNA和HBV抗原成分的存在,但术后骨髓CD34+细胞和PBMCs中皆长期存在HBV DNA.含有HBV DNA的骨髓来源CD34+细胞可能是含有HBV DNA的PBMCs的来源,也可能是肝移植术后PMBCs内HBV DNA长期存在的原因,及以后导致HBV复发的潜在危险因素.然而,现在尚无有效方法可以完全清除这些残存病毒,术后长期的抗病毒药物预防措施是必要的.     

Expression of stem cell markers on mononuclear cells derived from heparinized cadaveric organ donors before and after disconnection from the respirator

We have attempted to evaluate the level of the earliest human hematopoietic cell marker expression (CD34, CD117, CD133, CD184) on cells obtained from heparinized cadaveric organ donors before and after disconnection from the respirator. Moreover, we compared various cell populations: (1) coexpressing CD34/CD117; (2) CD34/CD133; (3) highly enriched hematopoietic stem cells (CD34+CXCR4+CD45+); and (4) highly enriched tissue-committed stem cells (CD34+CXCR4+CD45-). Finally, we analyzed whether the level of hematopoietic stem cell marker expression depended on the age of the donor. The expression of the membrane receptors (CD34, CD45, CD117, CD133, CD184) was studied by flow cytometry. We observed that the proportion of mononuclear cells expressing these markers slightly decreased in bone marrow harvested after disconnection from the respirator compared with the samples obtained before disconnection. Moreover, the proportion of cells expressing CD117 antigen depended on age of the donor.     

Gene delivery using human cord blood-derived CD34+cells into inflamed glomeruli in NOD/SCID mice

BACKGROUND: Bone marrow reconstitution using genetically-modified hematopoietic stem cells has been reported to confer resistance to inflammation and prevent renal injury in glomerulonephritis. Although this strategy has potentials for clinical use, taking hematopoietic stem cells from bone marrow is highly stressful for patients. In this regard, umbilical cord blood may be a useful alternative and, therefore, we focused on their suitability as a source of hematopoietic stem cells for transplantation-based therapy for glomerulonephritis. METHODS: CD34+ cells were obtained from human umbilical cord blood, retrovirally transduced with human beta-glucuronidase (HBG) gene, and transplanted into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. After confirming the successful chimerism, these mice were treated with lipopolysaccharide (LPS), and local HBG expression in glomeruli was examined using immunohistochemical analysis, HBG bioassay, and Western blot analysis. RESULTS: Clonogenic assay showed that 88.4 +/- 5.9% burst-forming unit-erythroid (BFU-E), 79.7 +/- 11.4% in colony-forming unit-macrophage (CFU-M), and 81.1 +/- 14.1% in colony-forming unit-granulocyte (CFU-G), respectively, possessed the transgene after transfection, suggesting that precommited cells were susceptible to retroviral infection. Flow cytometric analysis revealed that 24.1 +/- 14.5% of bone marrow cells in these chimera mice expressed human lymphocyte antigen (HLA) 8 weeks after transplantation. Also, clonogenic assay showed that a sustained engraftment of human hematopoietic cells expressed HBG. CD14-positive cells were recruited into the glomeruli upon LPS treatment and they secreted bioactive HBG, suggesting that cord blood-derived CD34+cells may differentiate into monocyte lineage while maintaining the expression of the transgene. CONCLUSION: These data indicate that umbilical cord blood cells can be utilized as a source of hematopoietic stem cells for the transplantation-based therapy of glomerulonephritis.     

粒细胞集落刺激因子和干细胞因子对骨髓单个核细胞的动员效率及其机制

目的 联合应用粒细胞集落刺激因子(G-CSF)和干细胞因子(SCF)动员骨髓单个核细胞,评价其动员效果,探讨CXCL12/CXCR4信号通路在骨髓单个核细胞动员中的作用及机制.方法 将昆明小鼠随机分为两组,治疗组皮下注射重组鼠G-CSF 100μg/(kg·d)和重组鼠SCF25μg/(kg·d),连续使用5d;对照组皮下注射等剂量的生理盐水.每组于不同时间点取小鼠骨髓,分离培养骨髓单个核细胞,计数成纤维样细胞集落形成单位(CFU-F)的个数;应用流式细胞仪分选CD34+ CXCR4+单个核细胞(MNCs);应用酶联免疫吸附试验(ELISA)法测定骨髓细胞外液CXCL12a的水平;采用逆转录-聚合酶链反应(RT-PCR)检测骨髓CXCL12 mRNA表达变化.结果 应用G-CSF/SCF后,骨髓及外周血中单个核细胞计数较对照组明显增加(P<0.01),CFU-F形成能力显著增强(P<0.05);流式分选表明CD34+ CXCR4+细胞占骨髓CD34+单个核细胞总数的(44.6±8.7)%;RT-PCR和EUSA检测示骨髓CXCL12 mRNA表达下降,骨髓细胞外液CXCL12蛋白也显著下降,两者变化一致.结论 G-CSF/SCF可有效地诱导骨髓单个核细胞动员,其机制可能是通过破坏骨髓CXCL12/CXCR4信号通路平衡,下调CXCL12/CXCR4间相互作用,以促进骨髓单个核细胞动员.     

Involvement of multiple subpopulations of human bone marrow cells in the regulation of allogeneic cellular immune responses

BACKGROUND: The identity of the cells in the human bone marrow that function as effective regulators of in vitro and possibly in vivo cellular immune responses is not well established. METHODS: Cell subpopulations were isolated from cadaver donor vertebral-body bone marrow cells (DBMC) by using immuno-magnetic microbeads and were tested as inhibitors (modulators) in cell-mediated lympholysis (CML) and mixed lymphocyte reaction (MLR) responses of normal peripheral blood lymphocytes stimulated with irradiated cadaver donor spleen cells. RESULTS: Compared with spleen cells as controls, un-irradiated T-cell depleted DBMC inhibited both the MLR and CML responses of allogeneic responder cells in a dose dependent manner (as in our previous reports). The inhibition was also mediated by a number of purified subpopulations including pluripotent CD34+ stem cells, and their CD34 negative early progeny of both lymphoid and myeloid lineages. These included DBMC enriched for non-T-cell lymphoid precursors (NT-LP/DBMC; i.e., DBMC depleted of CD3, CD15, and glycophorin-A positive cells) and DBMC positively selected for CD38+, CD2+, CD5+, and CD1+ lymphoid cells (all were depleted of CD3+ cells) as well as CD33+ (but CD15 negative) myeloid precursors. However, positively selected CD19+ B-cells and CD15+ myeloid cells did not inhibit the MLR and CML responses. The NT-LP/DBMC that had been repeatedly stimulated with irradiated allogeneic peripheral blood lymphocytes caused the strongest inhibition of the MLR and CML responses of the same allogeneic cells with 200 times fewer modulator cells needed than uncultured DBMC (P<0.001). Flow cytometric analysis revealed that majority of cells in these cell lines had become CD3+ TcR-alphabeta+ CD4+ and CD28+ cells. CONCLUSION: A variety of less differentiated cells of various lineages residing in the human bone marrow are immunoregulatory in vitro. Among them, there is at least one subset that can undergo differentiation in vitro into regulatory T cells that can be maintained in long-term cultures.     

由脐血单个核细胞和富集的CD34+细胞扩增的造血干/祖细胞的生理功能比较

目的 探讨由脐血单个核细胞(MNC)和富集的CD34+细胞起始扩增所得的造血干/祖细胞在体内植入及造血重建的能力.方法 从人脐血中分离出MNC和CD34+细胞,在体外扩增7 d.将非肥胖糖尿病型重症联合免疫缺陷型(NOD/SCID)小鼠分为四组,在接受亚致死剂量铯源照射后,进行细胞移植,实验A组接受由MNC培养得到的CD34+细胞和CD34-细胞;实验B组接受由富集的CD34+细胞培养得到的CD34+细胞和CD34-细胞;阳性对照组接受从脐血新鲜分离的CD34+细胞和CD34-细胞;阴性对照组不接受细胞移植,仅输注相同体积的IMDM培养基.6周后处死存活的小鼠,取其骨髓、脾脏和外周血细胞,分别进行细胞表型分析、集落和人特异性基因的检测.结果 经过体外扩增,以富集的CD34+细胞起始培养者的细胞总扩增倍数为39.8倍,远高于以MNC为起始细胞者的1.88倍.移植6周后,所有接受细胞移植的小鼠均存活,存活小鼠的骨髓和脾脏细胞中均能检测到人源细胞(CD45+细胞)及人源的各系血细胞,实验A组各类细胞的含量稍高于实验B组,且接受细胞移植小鼠的骨髓和脾脏细胞中可检测出人特异的Alu序列.结论 与从脐血中新鲜分离的细胞相比,扩增后的造血干/祖细胞的体内植入能力有所下降,以MNC起始扩增的造血干/祖细胞体内植入能力优于以富集的CD34+细胞起始扩增者,但二者体内造血重建能力的差异不显著.     

No evidence of hematopoietic stem cell mobilization in patients submitted to hepatectomy or in patients with acute on chronic liver failure

BACKGROUND: Liver regeneration is a heterogeneous phenomenon involving the proliferation of different cell lineages in response to injury. Under a strong positive selection pressure bone marrow derived stem cells may be involved in this process, by making a contribution to both parenchymal restoration and endothelial cell replacement. We investigate bone marrow stem cell migration to the liver in patients undergoing hepatectomy or with acute on chronic liver failure. METHODS: We enrolled 6 patients submitted to hepatectomy, 6 patients to cholecystectomy and 8 patients with acute decompensation of liver cirrhosis. Mobilization of CD34+ cells was evaluated by cytofluorimetry on peripheral blood samples at different time points; baseline, 1, 3, 7, 15 and 30 days after surgery and at admission, 1, 7 and discharge among patients with acute on chronic liver failure. 10 healthy subjects undergoing blood donation were also enrolled to evaluated the basal value of CD34+ cells. RESULTS: White blood cell counts remained in the normal range (4.1-9.8 x 10(9)/L) in all groups throughout the follow-up. In all patients of Groups 1, 2 and 3, circulating CD34+ failed to show statistically significant differences both as the absolute number and as the percentage at any time point compared to healthy controls. CONCLUSIONS: Bone marrow derived cell mobilization can not be detected after hepatectomy or during an acute decompensation on a cirrhotic liver. Under these circumstances liver regeneration can probably call upon mature hepatocytes and endogenous progenitor cells. The involvement of extrahepatic progenitors if any, is a rare and limited phenomenon.     

Correction of fanconi anemia Type C phenotypic abnormalities using a clinically suitable retroviral vector infection protocol

Fanconi anemia (FA) is a complex autosomal recessive disease with hematologic manifestations characterized by a progressive hypoplastic anemia, hypersensitivity to clastogenic agents, and an increased incidence of acute myelogenous leukemia. The cDNA that corrects one of four FA complementation subtypes, named Fanconi anemia Type C (FAC) has recently been identified. We constructed a simplified recombinant retrovirus (vMFGFAC) encoding only the FAC cDNA, and tested its ability to correct the FAC defect in a lymphocytic cell line and primary mobilized blood progenitor cells. In addition, the gene transfer efficiency using a clinically applicable gene transfer protocol into normal primitive hematopoietic progenitor cells, high proliferating potential colony forming cells (HPP-CFC), derived from CD34+ purified cord blood cells was examined. The gene transfer efficiency was significantly enhanced when cells were transduced with supernatant while adherent to a 30/35 KD fragment of fibronectin, FN30/35, and was similar to efficiency obtained by coculture with retrovirus packaging cells. Transduction of an FAC deficient lymphoid cell line with vMFGFAC supernatant resulted in an enhanced cell viability, and G-CSF mobilized peripheral blood cells from an FAC-deficient patient transduced with the vMFGFAC virus demonstrated enhanced progenitor cell colony formation. These data indicate that the vMFGFAC virus allows functional complementation of FAC in lymphoblasts and primary hematopoietic progenitors, and that primitive cord blood hematopoietic stem/progenitor cells can be transduced at an efficiency comparable to protocols using cocultivation if adherent to FN 30/35 fragment.